Millet alpha-amylase as well as encoding gene and application thereof

A technology of α-amylase and millet, which is applied in the fields of plant molecular biology and genetic engineering, can solve the problems of reducing pollen energy metabolism, insufficient starch accumulation, and few reports, so as to save the artificial detasseling step and maintain and reproduction, gene expression level precise effect

Active Publication Date: 2017-03-08
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the contrary, if amylase is overexpressed or silenced during pollen formation, it will reduce the level of pollen energy metabolism, resulting in insufficient starch accumulation, resulting in abortive pollen
However, there are few reports on rice pollen abortion genes.

Method used

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  • Millet alpha-amylase as well as encoding gene and application thereof
  • Millet alpha-amylase as well as encoding gene and application thereof
  • Millet alpha-amylase as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 Acquisition of millet α-amylase gene

[0051] 1. Extraction of millet RNA

[0052] Use the Biozol Reagent method to extract millet RNA: weigh 0.1g of fresh millet ear tissue and mix it with 1ml BiozolReagent, and let it stand at room temperature for 5 minutes; add 0.2ml chloroform for every 1ml Biozol Reagent, shake vigorously for 15s, and wait until the solution is fully emulsified, then Let stand at room temperature for 5 minutes, centrifuge at 12000rmp, 4°C for 15 minutes; carefully take out the centrifuge tube from the centrifuge, absorb the supernatant and transfer it to another new centrifuge tube; add an equal volume of isopropanol to the supernatant, and turn the centrifuge tube upside down After fully mixing, let stand at room temperature for 10min, centrifuge at 12000rmp and 4°C for 10min; discard the supernatant, a white precipitate may appear on the tube wall, add 0.5ml of 75% ethanol (prepared with sterilized DEPC water) to wash, invert and mix...

Embodiment 2

[0071] Example 2 Constructing the recombinant expression vector DX2182-XMAA of millet α-amylase

[0072] See the build process figure 1 , the vector DX2182 has pre-constructed the male gamete-preferred promoter PG47 with a sequence length of 2732 bp shown in SEQ ID No.3, and the transduction peptide with a sequence length of 216 bp shown in SEQ ID No.2. The amplified product of Example 1 was inserted into the Mlu1 and Sac1 restriction sites of the DX2182 vector, that is, the male gamete-preferred promoter and the downstream of the transduction peptide.

[0073] Primers SEQ ID NO:5-6 amplify the PCR product and recover a product of about 1300bp by 1% agarose gel electrophoresis. The vector DX2182 was digested with Mlu1 and Sac1, and the linearized vector was recovered.

[0074] 2X ligation kit connects the abortion gene to DX2182, the 10ul system is as follows:

[0075] 2.5 μl α-amylase PCR product (50ng), 2.5 μl enzyme-cut vector (100ng), 5 μl Ligation Mix, ligation procedu...

Embodiment 3

[0077] The acquisition of embodiment 3 transgenic rice plants

[0078] Agrobacterium EHA105 stored at -70°C was streaked on a plate containing Kan (50 μg / ml) + Rif (25 μg / ml) + streptomycin (50 μg / ml) and cultured at 28°C. Pick a single colony and inoculate it in 50ml of YEB liquid medium, shake it at 220rpm at 28°C for 12-16hr. Take 2ml of the bacterial liquid and transfer it to 100ml (containing antibiotics) YEP liquid medium, shake and culture at 28°C 220rpm to OD 600 = 0.5. Pre-cool on ice for 10 minutes, 5000rpm 10min (refrigerated centrifuge pre-cooled to 4°C). Wash twice with sterile deionized water (10ml each time), wash once with 10% glycerol and dissolve in 3ml 10% glycerol. Take 100 μl of competent cells and add 1 μl of the DX2182-XMAA plasmid obtained in Example 2, and transform by electroporation at 2.5KV. Culture at 28°C on a YEP culture plate containing kanamycin and rifampicin, select positive clones, and verify by PCR with DX2182-XMAA vector-specific prime...

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Abstract

The invention provides millet alpha-amylase as well as an encoding gene and application thereof, belonging to the technical field of gene engineering. An amino acid sequence of the millet alpha-amylase is shown by SEQ ID No.4 or is an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the sequence and has the same function, and a gene encoded nucleotide sequence of the millet alpha-amylase is shown by SEQ ID No.1 or a sequence with 80% of homology with this sequence. Under the driving of a pollen specific promoter, the millet alpha-amylase provided by the invention degrades starch in advance in a pollen developmental phase, germination energy for pollen cannot be provided, and the germination of the pollen is restrained, resulting in male abortion of crops. The millet alpha-amylase provided by the invention can be used for keeping the homozygosis recessive state of a male sterile plant, and meanwhile, a step of artificial stamen removal during hybrid seed production is saved, so that the millet alpha-amylase has broad application prospect in the aspects of crop germplasm resource improvement and genetic breeding.

Description

technical field [0001] The invention belongs to the technical fields of plant molecular biology and genetic engineering, and specifically relates to the application of millet α-amylase in causing pollen abortion, and also relates to the combination of the application and the use of modern biotechnology to apply it to a hybrid seed production technology system to ensure Seed production quality, and improve seed production efficiency; can also be applied to prevent the spread of transgenes. Background technique [0002] Utilization of plant heterosis is one of the most economical and effective ways to increase grain production, but there are still some limiting factors, such as the difficulty of combining good and bad crops, and the small proportion of hybrid crops. Although almost all crops have exploited heterosis to the greatest possible extent, there is still enormous potential. Therefore, many scholars have conducted research, especially in rice, the main way of heterosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/32C12N15/82A01H4/00A01H5/00
CPCA01H4/00C12N9/2422C12N15/8289C12Y302/01001
Inventor 黄培劲张维金雄霞陈思兰吴永忠
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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